RESUMO
Previously, we discovered that deletion of c-Rel in the Eµ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Eµ-Myc/cRel-/- cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Eµ-Myc/cRel-/- lymphomas highly resemble wild-type (WT) Eµ-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Eµ-Myc/cRel-/- lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Eµ-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours.
Assuntos
Linfoma , Proteínas Proto-Oncogênicas c-myc , Aminopiridinas , Animais , Enzimas Desubiquitinantes , Linfoma/metabolismo , Linfoma/patologia , Camundongos , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , PirimidinasRESUMO
Human milk oligosaccharides (HMOs) and their most abundant component, 2'-Fucosyllactose (2'-FL), are known to be immunomodulatory. Previously, it was shown that HMOs and 2'-FL bind to the C-type lectin receptor DC-SIGN. Here we show, using a ligand-receptor competition assay, that a whole mixture of HMOs from pooled human milk (HMOS) and 2'-FL inhibit the binding of the carbohydrate-binding receptor DC-SIGN to its prototypical ligands, fucose and the oligosaccharide Lewis-B, (Leb) in a dose-dependent way. Interestingly, such inhibition by HMOS and 2'-FL was not detected for another C-type lectin, langerin, which is evolutionarily similar to DC-SIGN. The cell-ligand competition assay using DC-SIGN expressing cells confirmed that 2'-FL inhibits the binding of DC-SIGN to Leb. Molecular dynamic (MD) simulations show that 2'-FL exists in a preorganized bioactive conformation before binding to DC-SIGN and this conformation is retained after binding to DC-SIGN. Leb has more flexible conformations and utilizes two binding modes, which operate one at a time via its two fucoses to bind to DC-SIGN. Our hypothesis is that 2'-FL may have a reduced entropic penalty due to its preorganized state, compared to Leb, and it has a lower binding enthalpy, suggesting a better binding to DC-SIGN. Thus, due to the better binding to DC-SIGN, 2'-FL may replace Leb from its binding pocket in DC-SIGN. The MD simulations also showed that 2'-FL does not bind to langerin. Our studies confirm 2'-FL as a specific ligand for DC-SIGN and suggest that 2'-FL can replace other DC-SIGN ligands from its binding pocket during the ligand-receptor interactions in possible immunomodulatory processes.
Assuntos
Lectinas Tipo C , Leite Humano , Trissacarídeos , Humanos , Fucose/análise , Lectinas Tipo C/metabolismo , Ligantes , Leite Humano/metabolismo , Receptores de Superfície Celular/metabolismo , Trissacarídeos/farmacologiaRESUMO
Hepatic iron is known to regulate insulin signaling pathways and to influence insulin sensitivity in insulin resistance (IR) patients. However, the role of insulin on hepatic iron homeostasis remains unexplored. Here, we report that insulin promotes transferrin-bound iron uptake but shows no influence on non transferrin-bound iron uptake in human hepatic HepG2 cells. As a mechanism we detected increased transferrin receptor-1 (TfR1) expression both at protein and mRNA levels. Unaltered stability of protein and transcript of TfR1 suggested the regulation at transcriptional level that was confirmed by promoter activity. Involvement of transcription factor hypoxia inducible factor-1 (HIF-1) was shown by mutational analyses of the TfR1 promoter region and by electrophoretic mobility shift assay. When HepG2 cells were transfected with specific siRNA targeted to 3'UTR of HIF-1α, the regulatory subunit of HIF-1; insulin-induced TfR1 expression and iron uptake were inhibited. Transfection of cDNA expressing stable form of HIF-1α reversed the increased TfR1 expression and iron uptake. These results suggest a novel role of insulin in hepatic iron uptake by a HIF-1 dependent transcriptional regulation of TfR1.
Assuntos
Antígenos CD/genética , Hepatócitos/metabolismo , Fator 1 Induzível por Hipóxia/fisiologia , Insulina/fisiologia , Ferro/metabolismo , Receptores da Transferrina/genética , Transcrição Gênica/fisiologia , Regiões 3' não Traduzidas , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized ß-3'galactosyllactose (ß-3'GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or ß-3'GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized ß-3'GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by ß-3'GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized ß-3'GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.
Assuntos
Células Epiteliais , Leucócitos Mononucleares , Galectinas/farmacologia , Células HT29 , Humanos , Oligossacarídeos/farmacologiaRESUMO
[structure: see text] An artificial macrocyclic fluorescent receptor 1 has been designed and synthesized for the recognition of urea. 1 shows significant fluorescence quenching on complexation with urea and thiourea in chloroform and thus may be used as a synthetic fluorescent molecular sensor for their determination in a nondegradative way.
Assuntos
Clorofórmio/química , Corantes Fluorescentes/síntese química , Ureia/análise , Sítios de Ligação , Corantes Fluorescentes/química , Conformação Molecular , Estrutura Molecular , Espectrometria de Fluorescência , Ureia/químicaRESUMO
The syntheses in good yields of some new difunctionalized 1,8-naphthyridines 4, 6, 8 and 9 and a novel triethylene glycol ether-linked dinaphthyridine, 10a, along with the mononaphthyridine-linked ether alcohol 10b are described. An improved and milder method for the synthesis of 2,7-diamino-1,8-naphthyridine (14) is also reported.
Assuntos
Naftiridinas/síntese química , Naftiridinas/química , Polietilenoglicóis/química , TemperaturaRESUMO
Oxygen sensing transcription factor HIF-1 is activated due to accumulation of regulatory subunit HIF-1α by posttranslational stability mechanism during hypoxia or by several other stimuli even in normoxia. HIF-1α is also regulated by NF-kB mediated transcription mechanism. Reactive oxygen species (ROS) act as an important regulator of HIF-1 either by affecting prolyl hydroxylase activity, the critical determinant of HIF-1α stabilization or by activating NF-kB to promote HIF-1α transcription. Insulin is known to activate HIF-1 by a ROS dependent mechanism but the molecular mechanism of HIF-1α regulation is not known so far. Here we show that insulin regulates HIF-1α by a novel transcriptional mechanism by a ROS-sensitive activation of Sp1 in 3T3-L1 preadipocyte. Insulin shows little effect on HIF-1α protein stability, but increases HIF-1α promoter activity. Mutation analyses, electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirm the role of Sp1 in HIF-1α transcription. We further demonstrate that insulin-induced ROS generation initiates signaling pathway involving phosphatidylinositol 3-kinase and protein kinase C for Sp1 mediated HIF-1α transcription. In summary, we reveal that insulin regulates HIF-1α by a novel transcriptional mechanism involving Sp1.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Insulina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células 3T3-L1 , Região 5'-Flanqueadora , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , Elementos de Resposta , Deleção de SequênciaRESUMO
Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring beta-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound beta-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026. The inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.